There is a fundamental gap in understanding pertaining to why standard fluid resuscitation protocols work so poorly in alcohol-intoxicated trauma victims with hemorrhagic shock, representing a significant portion of all trauma cases. Continued existence of this gap represents an important problem, because until it is filled, the development of effective therapies to prevent systemic inflammation and prolonged hypotension in trauma patients will continue to be unsuccessful. The long-term goal of this study is to develop effective therapies to reduce elevated microvascular permeability and edema. The overall objective in this application is to determine the effectiveness of the bioactive lipid sphingosine-1-phosphate (S1P) for ameliorating alcohol-induced elevated microvascular permeability. The central hypothesis is that S1P reduces alcohol/injury-induced microvascular hyperpermeability by activating S1P receptor-1 (S1P1R), causing initial junctional repair, and longer term pro-survival signals, thereby promoting a healthy endothelial barrier. The rationale for the proposed research is that it will provide important details obtained from a relevant model of combined alcohol intoxication and hemorrhagic injury that will be needed for development of effective fluid resuscitation strategies. Guided by strong preliminary data, the central hypothesis will be tested with three specific aims: Aim 1 is to determine the optimal dose and window of opportunity for S1P therapy after combined alcohol intoxication and hemorrhagic shock. Aim 2 is to test selective S1P1R agonist therapies to ameliorate alcohol/shock-induced microvascular hyperpermeability and hypotension. Aim 3 is to determine the extent to which S1P-induced, RhoA-mediated junctional enhancement and cell survival signals contribute to ameliorate endothelial barrier dysfunction. These aims will utilize an established conscious rat model of combined alcohol intoxication and hemorrhagic shock and resuscitation. The experimental endpoints will be the extent to which S1P or S1P1R agonists ameliorate hypotension, microvascular leakage in the gut, and gut barrier injury. The in vivo studies will be complemented by an investigation of the ability of S1P or S1P1R agonists to improve barrier integrity of cultured microvascular endothelial cell monolayers exposed to alcohol. Pharmacologic antagonists or specific siRNA knockdown will be used to assess the role of the S1P1R. A combination of intravital microscopy, laser confocal microscopy, and biochemical analyses will be used to study microvascular endothelial junctional architecture and survival signals in individual endothelial cells of postcapillary venules. The contribution of the proposed research will be significant because it will provide proof of concept from a rodent model that alcohol-induced amplifications of elevated microvascular permeability contribute to the lack of success with current clinical interventions used to resuscitate alcohol-intoxicated hemorrhagic shock patients. The proposed research is innovative because it represents a substantive departure from the status quo by investigating a therapeutic approach to ameliorate alcohol intoxication-induced microvascular hyperpermeability as the key approach to stabilize blood pressure.